Single-stranded DNA is useful in a number of important technologies. For example, the determination of the sequence of a nucleic acid has had enormous impact in the fields of molecular biology, biochemistry and genetics. Many sequencing methods require the use or generation of single-stranded DNA at some stage in the methods. Nucleic acid sequencing has promoted considerable research and commercial activity. A single nucleic acid is generally characterized by nucleotide sequence, molecular weight, size and shape. A useful sequencing procedure is described, for example, by Gyllenstein et al (Proc.Natl.Acad.Sci.USA, 85, p. 7652-7656, October, 1988).
Single-stranded nucleic acids are also useful as probes in medical diagnostics based on the hybridization of complementary nucleotides of nucleic acids to detect pathogens, diseases, genetic features or disease-causing situations. The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions which disrupt the hydrogen bonding between complementary nucleotides.
In addition, hybridization assays are used in forensic testing, both criminal (identifying persons by samples of hair, blood or semen left at the scene of the crime or on the victim) and non-criminal (such as for paternity determinations and immigration screening).
The use of complementary nucleic acids to detect small quantities of targeted nucleic acids has been enhanced considerably by the development of polymerase chain reaction procedures as described, for example, in U.S. Pat. No. 4,683,195 (issued Jul. 28, 1987 to Mullis) and U.S. Pat. No. 4,683,202 (issued Jul. 28, 1987 to Mullis et al). Without going into detail regarding these procedures, they involve the use of a polymerase to make extension products which are complementary to the target nucleic acid strands, which extension products are denatured and multiplied themselves many times in cyclical fashion. Thus, the original targeted strands are multiplied greatly for eventual detection. In some instances, one or both primers used in such procedures are biotinylated for complexation with avidin for capture or detection.
Nucleic acids have been labeled with a number of detectable moieties in order to facilitate the detection of hybridized strands. Biotin has been commonly used as a detectable moiety because it readily combines with avidin to form stable detectable complexes.
The high affinity of biotin for the glycoprotein avidin provides the basis for many established procedures for the detection and isolation of biotin-associated proteins or nucleic acids. With the introduction of biotinylated nucleotide analogs by various researchers, it has become possible to apply this affinity system to the detection of specific nucleic acids by using biotinylated hybridization probes. The strong avidin-biotin complex can provide a single-step, high yield retrieval procedure of targeted nucleic acids from crude mixtures.
Because workers in the art have wanted to avoid harsh chemical denaturing conditions (for example, 6 molar guanidine HCl, pH 1.5) to break avidin-biotin complexes, many reagents have been synthesized to enable the captured nucleic acid to be released from the capture probe and isolated. For example, in some instances, a nucleic acid is chemically modified to have a chemically cleavable linkage between the nucleotides and the biotin. Shimkus et al (Proc.Natl.Acad.Sci.USA, 82, pp. 2593-2597, 1985) describe the use of a disulfide bond as a means for reversibly binding nucleotides to avidin-agarose columns. Other chemically cleavable nucleotides are described in U.S. Pat. No. 4,772,691 (issued Sep. 20, 1988 to Herman) to provide a means for chemical cleavage under relatively mild conditions.
While the chemically-modified reagents known in the art serve the desired function, it would be highly desirable to avoid the need to synthesize or purchase such reagents due to the tedious synthetic procedures and expense. It would be desirable to have a simple, but effective means for isolating or preparing biotinylated nucleic acids without harsh chemical denaturing conditions, and the need for expensive reagents.